The goals of this project are to bring new biomarkers to the clinic that can transform decision-making for: 1) the post-surgical management of cutaneous melanoma patients with stages I, II, & III (i.e. loco-regional) disease; and 2) the surveillance of patients with stage IV metastatic melanoma lacking BRAFV600 or NRASQ61 mutations. Using SNaPshot and allele-specific PCR assays that identify mutations in the TERT promoter, and in BRAF and NRAS, we have generated promising preliminary data demonstrating that TERT promoter mutations are common in primary and metastatic melanoma, and are significantly associated with BRAF or NRAS mutations. These data support recent findings that TERT promoter mutations are also associated with adverse survival outcomes in several cancers including melanoma. This application is responding to PA-12-014: Validation of Molecular Diagnostics to Predict Patient Outcomes Using Specimens from Multi-Site Cancer Trials. As such, it is a pilot-scale validation study to prove the clinical usefulnessof a combined BRAF/NRAS/TERT SNaPshot assay in tumors for patients with stages I, II, & III disease, and a TERT qPCR assay of the circulating tumor DNA from patients with stage IV disease. The specific goals of this project are to define the sensitivity and specificity of the tumor-based assays to predict the development of metastatic disease in patients with stages I, II and III melanoma; and for the plasma-based assay, to predict treatment response and disease progression in patients with stage IV melanoma lacking BRAFV600 or NRASQ61 mutations. To achieve our goals we will analyze a large group of melanoma patients with stages I-IV disease and complete clinical follow up data prospectively accrued into the NYU Melanoma Biorepository. In Aim 1 we will analyze tumor specimens from patients with stages I-III melanoma to determine the degree to which TERT promoter mutations are associated with survival endpoints and whether the association is influenced by the presence of BRAF or NRAS mutations, or a newly described germline polymorphism in the TERT promoter. In Aim 2, we will study circulating tumor DNA from stage IV melanoma patients undergoing treatment with immune checkpoint blockade whose tumors lack BRAFV600 or NRASQ61 mutations. We will determine the sensitivity of our TERT mutant-specific qPCR assay to predict treatment response and disease progression in comparison to radiographic scans. In other studies, we have developed blood-based disease monitoring assays for patients whose tumors have either BRAFV600 or NRASQ61 mutations; for patients whose tumors lack these mutations there is no clinically useful blood-based marker, so new assays are needed. The proposed studies, therefore, have the potential to lay the groundwork for new biomarkers to fundamentally improve the management of melanoma patients with: 1) localized disease, by identifying those with the greatest need for adjuvant therapy, and 2) metastatic disease, by helping to detect disease progression ahead of radiographic scans so that therapies can be modified at the earliest signs of recurrence.